The objective of the proposed research is to investigate the biochemical properties, inborn errors and genetic regulation of several enzymes involved in heme biosynthesis. Human gamma-aminolevulinic acid dehydratase (ALAD) and uroporphyrinogen-I-Synthetase (UROS) will be purified to homogeneity from human erythrocytes. Immobilized ALAD will be used to synthesize gram quantities of porphobilinogen (PBG), the UROS substrate. PBG availability will permit isolation and characterization of UROS by the development of efficient purification procedures and sensitive electrophoretic techniques. The purified ALAD and UROS enzymes will be used for 1) physical and kinetic studies, 2) antibody production, and 3) coupled microassays of ALAD and gamma-aminolevulinate synthetase (ALAS). Studies of the molecular pathology in acute intermittent porphyria (AIP) will include 1) characterization of UROS isozymes and their modulation during remission and acute attacks and 2) determination of the quantity of immunologically cross-reacting material (CRM) in erythrocytes and cultured cells from patients with this dominantly inherited disease. Similarly, the nature of the ALAD deficiency in a newly discovered, dominantly inherited, human ALAD variant will be investigated. Somatic cell genetic techniques will be used to study the genetic regulation of heme biosynthesis. The chromosomal localization of genes controlling the synthesis of ALAS, ALAD, and UROS in man will be investigated in human-rodent somatic cell hybrids. Electrophoretic techniques will be developed to discriminate the respective human and rodent isozymes. The temporal expression of ALAS, ALAD, and UROS will be assessed in an inducible human cell line. These studies should provide increased understanding of the molecular pathology of these dominantly inherited defects and the genetic regulation of human heme biosynthesis.